Enzymes are biological catalysts. They are proteins and are commonly water-soluble. Enzymes are isolated and used in an extraordinarily large and diverse number of commercial applications, including analytical, medical, food-processing, and industrial applications. For example, enzymes are used to prepare food products such as cheese, bread, and alcoholic beverages; enzymes are used to resolve amino acids; enzymes are used in meat tenderizers, detergent formulations, leather tanning agents, and in digestive aids. Enzymes are also used extensively in the processing of starch, such as in starch hydrolysis, sucrose inversion, glucose isomerization, etc. These uses for enzymes, as well as many others, are addressed in great length in the relevant literature.
Enzymes in solution are difficult to recycle while maintaining high catalytic activity. Even without attempting to recycle the enzymes, it is often difficult to maintain high catalytic activity of enzymes for any extended period. These factors often make enzymatic catalysis an expensive proposition due to the necessity to replace the enzyme often.
To minimize the need for replacement, enzymes have been immobilized or otherwise insolubilized on inert supports or carriers. The enzyme remains catalytically active, but because the enzyme is affixed to a solid support, it can be removed from the reaction solution by filtering or screening. By immobilizing the enzyme on a solid support, the enzyme can be recycled more easily and the active useful life of each enzyme batch can be increased.
Immobilized enzymes are used in many different reactor systems, such as in packed columns, stirred tank reactors, fluidized-bed reactors, etc. In general, immobilizing the enzyme provides one or more benefits, including more favorable conditions wherein the enzyme can be used, greater structural stability, increased active life span of the enzyme, minimized effluent problems, minimized material handling problems, and (potentially) increased activity of the enzyme itself.
The patent literature describes a great many means of immobilizing enzymes on an inert support. One general method is to adsorb the enzyme at a solid surface as, for example, when an enzyme such as amino acid acylase is adsorbed on a cellulosic derivative such as DEAE-cellulose; papain or ribonuclease is adsorbed on porous glass; catalase is adsorbed on charcoal; trypsin is adsorbed on quartz glass or cellulose, chymotrypsin is adsorbed on kaolinite, etc.
Another general method to immobilize enzymes is to trap an enzyme in a gel lattice, such as glucose oxidase, urease, papain, etc., being entrapped in a polyacrylamide gel; acetyl cholinesterase being entrapped in a starch gel or a silicone polymer; glutamic-pyruvic transaminase being entrapped in a polyamide or cellulose acetate gel, etc.
A further general method is to use a cross-linking reagent to bind the enzyme to the support. In this approach, bifunctional or polyfunctional reagents that induce intermolecular cross-linking covalently bind the enzymes to the solid support. Glutaraldehyde or bisdiazobenzidine-2,2′-disulfonic acid are conventionally used as cross-linking reagents.
Conventional methods of immobilizing enzymes, however, all possess distinct drawbacks that detract from their use in industrial processes. For example, when an enzyme is directly adsorbed on the surface of a support, the binding forces that result between the enzyme and the support are often quite weak. Consequently, the enzyme is often readily desorbed from the support. Alternatively, the enzyme may be deactivated partially or extensively once immobilized (presumably due to conformational constraints caused by the binding reaction or due to adverse interactions between the support and the active site of the enzyme).